Download Environmental bacteriophages: viruses of microbes in aquatic

REVIEW ARTICLE
published: 24 July 2014
doi: 10.3389/fmicb.2014.00355
Environmental bacteriophages: viruses of microbes in
aquatic ecosystems
Télesphore Sime-Ngando*
Laboratoire Microorganismes: Génome et Environnement, UMR CNRS 6023, Clermont Université Blaise Pascal, Aubière, France
Edited by:
David Georges Biron, Centre National
de la Recherche Scientifique, France
Reviewed by:
Anne-Claire Baudoux, Centre National
de la Recherche Scientifique, France
Herve Moreau, Centre National de la
Recherche Scientifique, France
*Correspondence:
Télesphore Sime-Ngando, Laboratoire
Microorganismes: Génome et
Environnement, UMR CNRS 6023,
Clermont Université Blaise Pascal,
BP 80026, 24 Avenue des Landais,
Aubière 63171, France
e-mail: telesphore.sime-ngando
@univ-bpclermont.fr
Since the discovery 2–3 decades ago that viruses of microbes are abundant in marine
ecosystems, viral ecology has grown increasingly to reach the status of a full scientific
discipline in environmental sciences. A dedicated ISVM society, the International Society
for Viruses of Microorganisms, (http://www.isvm.org/) was recently launched. Increasing
studies in viral ecology are sources of novel knowledge related to the biodiversity of living
things, the functioning of ecosystems, and the evolution of the cellular world. This is
because viruses are perhaps the most diverse, abundant, and ubiquitous biological entities
in the biosphere, although local environmental conditions enrich for certain viral types
through selective pressure. They exhibit various lifestyles that intimately depend on the
deep-cellular mechanisms, and are ultimately replicated by members of all three domains
of cellular life (Bacteria, Eukarya, Archaea), as well as by giant viruses of some eukaryotic
cells.This establishes viral parasites as microbial killers but also as cell partners or metabolic
manipulators in microbial ecology. The present chapter sought to review the literature on
the diversity and functional roles of viruses of microbes in environmental microbiology,
focusing primarily on prokaryotic viruses (i.e., phages) in aquatic ecosystems, which form
the bulk of our knowledge in modern environmental viral ecology.
Keywords: aquatic ecosystems, viruses, lysis, lysogeny, bacteria, horizontal gene transfers, food web dynamics,
biogeochemical cycling
INTRODUCTION
With the discovery few decades ago that viral parasites of microbes
are abundant in marine ecosystems (Torrella and Morita, 1979;
Bergh et al., 1989), aquatic viral ecology has increasingly grew
to reach the status of full scientific discipline in environmental sciences, with the recent launch of a dedicated ISVM society,
i.e., the International Society for Viruses of Microorganisms1 . As
infectious agents of potentially all types of living cells, viruses
are the most abundant biological entities in the biosphere. They
are ubiquitous components of the microbial food web dynamics
in a great variety of environments, including the most extreme
ecosystems. Moreover, in spite of the difficulties to routinely
observe and describe biological nanoparticles, combined with the
absence of conserved evolution tracers such as RNA ribosomal
genes, we now consider that viruses represent the greatest reservoir of non-characterized genetic diversity and resources on the
earth (Suttle, 2007). They contain genes that code for essential
biological functions such as photosynthesis (Lindell et al., 2005),
making their hosts powerful vehicles for genetic exchanges in the
environment. Because lytic viruses killed their hosts, they play
fundamental roles in cycling nutrients and organic matter, structuring microbial food webs, governing microbial diversity and,
to a lesser extent, by being a potential food source for protists
(Sime-Ngando and Colombet, 2009). As symbionts, viruses can
also form long-lived association with their specific hosts, reducing their fitness, or allowing infected hosts to remain strong
1 http://www.isvm.org/
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competitors through mutualistic symbioses (Roossinck, 2011). In
addition, the discovery and characterization of the unique group
of archaeal viruses are influencing the field of prokaryotic virology, increasing our knowledge on viral diversity and changing
perspectives on early stages of evolution (cf. Prangishvili et al.,
2006).
Recent studies in aquatic viral ecology are source of novel
knowledge related to the biodiversity of living things, the functioning of ecosystems, and the evolution of the cellular world.
Viruses exhibit various life strategies that intimately depend on
the deep-cellular mechanisms, and are ultimately replicated by all
members of the three domains of cellular life: Bacteria, Eukarya,
and Archaea. This establishes viruses as microbial killers (i.e., bad
viruses) but also as cell partners and manipulators (i.e., good
viruses) in the world of aquatic ecosystem (Rohwer and Thurber,
2009; Roossinck, 2011). The present chapter sought to review
the literature on the diversity and functional roles of viruses in
aquatic microbiology, focusing on prokaryotic viruses (i.e., bacteriophages) which form the bulk of our knowledge in aquatic viral
ecology.
DEFINITION AND LIFESTYLES
Viruses are biological entities consisting of single- or doublestranded DNA or RNA surrounded by a protein and, for some
of them, a lipid coat. In aquatic systems, most viruses are
tailed or untailed phages, with a capsid diameter often smaller
than 250 nm, based on direct transmission electron microscopy
observation. Viruses have no intrinsic metabolism and need
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the intracellular machinery of a living and sensitive host cell
for all processes requiring energy. They have various life cycles,
all starting with diffusive passive fixation on specific receptors
(often transporter proteins) present at the surface of a host cell,
followed by injection of the viral genome into the host cell.
In the lytic cycle, the viral genome induces the synthesis of
viral constituents, including the replication of the viral genetic
material. A number of progeny viruses are then produced and
released into the environment by the fatal rupture of the host
cell.
In the chronic cycle, the progeny viruses are episodically
or constantly released from the host cell by budding or extrusion, without immediate lethal events. This cycle is less well
known in aquatic microbiology, but it is common in metazoan
viruses such as Herpes and Hepatitis viruses or rhabdoviruses.
Chronic viral infection is a dynamic and metastable equilibrium
process which ends with the lysis of the host cell after serial
budding of lipid membrane-coated viruses, as seen in hosts of
the marine protist Emiliania huxleyi (Mackinder et al., 2009).
Recently, chronic infection without host lysis has been reported
for the first time in the marine primary producer Ostreococcus tauri, where the low rate of viral release through budding
FIGURE 1 | Virus–microbe interactions range in a gradient from true
non-lethal parasitism (i.e., stable coexistence) to fatal lytic infection
(lysis), with intermediate mutualistic lifestyles (lysogeny and
pseudolysogeny). Because of the existence of such a large panel of
lifestyles and in conjunction with the fact that all types of cells are sensitive to
unique viruses, these biological entities are considered the most diverse,
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(1–3 viruses cell−1 day−1 ) allows cell recovery and the stable coexistence of viruses and their hosts (Thomas et al., 2011; Clerissi et al.,
2012).
In the lysogenic cycle, the viral genome integrates the genome
of the host cell and reproduces as a provirus (or prophage) until
an environmental stress to the immune host cell sets off a switch
to a lytic cycle. Both the provirus and the host cell benefit from
lysogeny. Lysogeny provides a means of persistence for viruses
when the abundance of the host cells is very low. Prophages may
affect the metabolic properties of host cells which can acquire
immunity to superinfections and new phenotypic characteristics
such as antibiotic resistance, antigenic changes, and virulence
factors, resulting in niche expansion for viral hosts (Figure 1).
A variant to the lysogenic cycle is the so-called carrier state or
pseudolysogenic cycle, where the viral genome is not integrated
with the host genome but rather remains in an “inactive state”
within the host cell. There is no replication of the viral genome
which is segregated unequally into progeny cells, most likely for
a few generations. Pseudolysogenic viruses probably occur in
very poor nutrient conditions where host cells are undergoing
starvation and cannot offer the energy necessary for viral gene
expression.
abundant, and ubiquitous biological entities in the biosphere where they have
tremendous effects on the diversity of living things, the functioning of
microbial ecosystems, and the evolution of the cellular world. Some of these
direct (solid lines) and indirect (dashed lines) effects on aquatic microbial
processes are highlighted in this figure. Please refer to the main text for
abbreviations.
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TAXONOMY
There are three domains of life – Archaea, Bacteria, and Eukarya –
that consist only of cellular organisms (Woese et al., 1990). Because
viruses lack the ribosomal RNA nucleotide sequence upon which
these cellular domains of life are based, they cannot be integrated
into the cellular tree of life (Breitbart et al., 2002; Rohwer and
Edwards, 2002), although susceptibility to virus infection is a common feature of all members of the three domains of life. In the
absence of universal evolution markers for the entire viral world,
viruses have been grouped by many different methods, according
to various criteria: the nature of the host, the characteristics of
the free virions (phenotype, genotype, resistance to organic solvents for viruses with lipid coat, etc.), or even the name of the
related illness, the laboratory or the researcher working on the
targeted viruses. The 9th report by the International Committee
on Taxonomy of viruses (ICTV)2 includes 6 orders, 87 families, 19
subfamilies, 349 genera, and 2284 virus and viroid species, defined
as a group of viruses that constitutes a replicative lineage and
occupies a particular ecological niche (Regenmortel, 1992). Hurst
(2011) introduced the idea that the taxonomy of viruses and their
relatives could be extended to the domain level, and suggests the
creation of an additional biological domain that would represent
the acellular infectious agents that possess nucleic acid genomes
or the genomic acellular agents. The proposed domain title is
Akamara, whose derivation from the Greek (a + kamara) would
translate as “without chamber” or “without void.” The domain is
divided into two kingdoms. The kingdom Eurivia includes true
viruses and those satellite viruses whose genomes code for their
own capsid proteins, and is separated in two Phyla (RNA and
DNA viruses). The second kingdom (Viroidia) forms one phylum
that includes viroids along with other groups of related agents
whose genomes likewise do not code for their structural “shell”
proteins.
A new way to classify phages has also been proposed based
on the complete sequences of 105 viral genomes. This so-called
phage proteomic tree places phages relative to their neighbors and
all other phages included in the analysis, which is a method that
can be used to predict aspects of phage biology and evolutionary
relationships, and to highlight genetic markers for diversity studies
(Rohwer and Edwards, 2002). The approach is useful for those
phages whose complete genomes have been deciphered (i.e., a
minority of environmental viruses); primarily those belonging to
a common pool of genes (e.g., following genetic recombination),
or which have evolved from a common ancestor. It is well known
that the genomes of some phages, if not most, are mosaics of
genes from various sources, including other phages and their hosts
(Hendrix et al., 1999).
PHENOTYPIC TRAITS
The first descriptions of the global diversity of viruses are from
the general forms of virus-like particles observed via transmission electron microscopy. In aquatic samples, viral phenotypes are
limited, mainly including tailed or untailed particles with capsid
heads, characteristics of bacteriophages. Tailed phages belong to
the order Caudovirales, all of which are double-stranded DNA
2 http://www.ictvonline.org/index.asp?bhcp=1
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Viruses of environmental microbes
viruses that generally represent 10–40% of the total abundance
of viruses in aquatic systems (see comparative tables in Wommack and Colwell, 2000; Sime-Ngando and Colombet, 2009).
Within Caudovirales, three families emerge as quantitatively dominant: Siphoviridae with long, non-contractile tails (e.g., Phage
lambda), Podoviridae with a short, non-contractile tail (e.g.,
Phage T7), and Myoviridae with contractile tails of variable length
(e.g., Phage T4). In most studies, non-tailed capsids dominate
viral abundances. This may be an artifact due to the effects of
mechanic shocks resulting from handling, primarily ultracentrifugation (Colombet et al., 2007), because 96% of the 5500
specimens of described bacteriophages are tailed particles (Ackermann, 2007). However, a recent global morphological analysis of
marine viruses suggested that non-tailed viruses, which comprised
50–90% of the viral particles observed, might represent the most
ecologically important component in natural viral communities
(Brum et al., 2013).
Phenotypic traits and viral morphs in aquatic viruses are
cryptic of the selective pressures faced by these communities, and provide insight into host range, viral replication and
function (Suttle, 2005). For instance, myoviruses are mostly
lytic with a large spectrum of sensitive hosts, which is a
competitive advantage that can be assimilated to r-strategist
species thriving with high proliferation rates in fluctuating environments. In contrast, podoviruses are more highly specific
to their hosts, with siphoviruses being intermediate between
myo- and podoviruses. In addition, several siphoviruses can
encode their genome into their hosts for several generations
(i.e., lysogeny), which can be rather assimilated to K-strategist
species, characteristics of stable environments. Combined with
the capacity of viruses to potentially face almost all types
of environments and the related interfaces (Hurst, 2011), the
ability of viruses to develop along the r-K-selection continuum, i.e., from typical r (e.g., prokaryotes) to K (e.g., vertebrates) strategists (Suttle, 2007), may help to explain their
ubiquity, hence the notion of the virosphere (i.e., viral biosphere).
GENOMIC DIVERSITY
ICTV-reported viral species are mostly known from their isolated
hosts in laboratory cultures which, in the case of environmental
samples, may not exceed 1% of the total prokaryotes (Hugenholtz et al., 1998). This implies that the diversity of environmental
viruses is huge, although the bulk of the estimated 1031 viruses
in the biosphere is unknown (Rohwer and Edwards, 2002). In
addition to whole genome sequencing of specific phages (Allen
et al., 2011) or genomes assembled from metagenomics datasets
(Rosario et al., 2009), molecular approaches applied to uncultured
complex communities are thus critical and offer windows to the
largest uncharacterized reservoir of diversity on the earth (Hambly
and Suttle, 2005). Polymerase chain reaction (PCR)-based methods are restricted to chosen viral groups as no gene is universally
conserved among viruses, while part of the existing diversity of
these viral groups is missed because PCR primers are based on previously identified sequences described in public databases. Viral
metagenomics gives access to an exhaustive view of uncultured
viral diversity (Breitbart et al., 2002), and has so far revealed an
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important unknown diversity and an unexpected richness of viral
communities (Edwards and Rohwer, 2005).
Despite the ecological importance of viruses, a fundamental
hindrance to their integration into microbial ecology studies is
the lack of suitable reference bacteriophage genomes in reference
databases. Currently, only eight bacterial phyla (Proteobacteria, Firmicutes, Bacteriodetes, Actinobacteria, Cyanobacteria,
Chlamydiae, Tenericutes, and Deinococcus-Thermus) of 29 phyla
with cultured isolates have sequenced phage representatives, contributing only 0.001% of genome sequence databases (Bibby,
2014). From these few phage genomes, comparative genomics
have revealed an impressive level of genomic diversity and novelties as well as hypotheses on potential adaptation of phage genome
to aquatic environments. For example, comparison of 26 T4-like
genomes of myoviruses infecting diverse marine cyanobacteria
(Prochlorococcus or Synechococcus) has revealed highly syntenic
hierarchical cores of genes, with DNA replication genes observed
in all genomes, followed by previously described, virion-structural
and various hypothetical genes. Beyond previously described
cyanophage-encoded photosynthetic and phosphate stress genes,
genes involved in various putative functions (e.g., phytanoylCoA dioxygenase, 2-oxoglutarate) were indeed described, as well
as non-core genes that may drive complex niche diversification (Sullivan et al., 2010). The unveiling of the first genome
of a deep-photic marine cyanobacterial siphovirus highlighted
the prevalence of lysogenic lifestyle and significant divergence
and size differences with previously sequenced siphoviruses,
and the absence of photosynthetic genes which have consistently been found in other marine cyanophages (Sullivan et al.,
2009). Similarly, the genomic and functional analysis of a novel
marine siphovirus of marine Vibrio revealed a larger genome
(80,598 bp) compared to that of most known siphoviruses,
with a novel shell symmetry that confers a remarkable stability to a variety of physical, chemical, and environmental factors
(Baudoux et al., 2012). Whole genome sequencing/reconstruction
of phages that are currently unrepresented in the database will
thus likely provides deep insights into and have a significant
impact on our view of viral diversity, ecology, and evolution,
while providing molecular tools for the study of groups of
viruses.
Whole genome comparisons indeed have also shown that there
are conserved genes shared among all members within certain viral
taxonomic groups. These conserved genes can be targeted using
PCR amplification and sequencing for diversity studies of groups
of cultured and environmental viruses. Examples of such genes
are structural proteins such as gp20, which codes for the capsid
formation in T4 phage-like viruses, DNA polymerases for T7-like
podophages, or the RNA-dependent RNA polymerase fragment,
which has been used to identify novel groups of marine picornaviruses (Culley et al., 2003). All of the conserved gene studies
suggest that environmental viral diversity is high and essentially
uncharacterized (Breitbart and Rohwer, 2005).
For the whole environmental communities, molecular fingerprinting approaches that separate PCR-generated DNA products,
such as denaturing gradient gel electrophoresis (DGGE, Short and
Suttle, 2002) and pulse-field gel electrophoresis (PFGE, Wommack et al., 1999), have been widely used but with limited results,
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restricted to double-stranded DNA viruses. With this approach,
the genome size of aquatic viruses fluctuates from 10 to about
900 kb, with mean ranges of 10–630 kb and 10–660 kb in marine
and freshwater systems, respectively. The frequencies of the distribution of genome size classes are multimodal, with peaks in the
interval < 70 kb and a mean at 50 kb.
An introduced fingerprinting approach adapted to viruses, randomly amplified polymorphic DNA-PCR (RAPD-PCR), allows
sampling of viruses at the genetic level without requiring viral isolation or previous sequence knowledge (Winget and Wommack,
2008). RAPD-PCR is accurate in assessing DNA viral richness in
water samples by using single 10-mer oligonucleotide primers to
produce amplicons (PCR-generated DNA fragments) and banding patterns, with each likely representing a single amplicon that
originates from viral template DNA. Such an approach has been
demonstrated to match observations from other community profiling techniques, revealing more temporal than spatial variability
in viroplankton assemblages. Hybridization probes and sequence
information can also be easily generated from single RAPDPCR products or whole reactions, providing a tool for routine
use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence
information.
Metagenomics has revolutionized microbiology by paving the
way for a culture-independent assessment and exploitation of
microbial and viral communities present in complex environments (Simon and Daniel, 2011). Metagenomic viral analyses
or virome studies suggest that environmental viral diversity is
high and essentially uncharacterized (Angly et al., 2006; Roux
et al., 2012). Metagenomic analyses of 184 viral assemblages collected over a decade and representing 68 sites in four major
oceanic regions showed that most of the viral DNA and protein sequences were not similar to those in the current databases
(Angly et al., 2006). Global diversity was very high, presumably
several hundred thousand species, and regional richness varied on
a North–South latitudinal gradient. However, most viral species
were found to be widespread, supporting the idea that marine
viruses are widely dispersed and that local environmental conditions enrich for certain viral types through selective pressure. A
study on comparative viral metagenomics highlighted that freshwater, marine, and hypersaline environments were separated from
each other despite the vast geographical distances between sample
locations within each of these biomes, suggesting a genetic similarity between viral communities of related environments (Roux
et al., 2012).
Interrogation of microbial metagenomic sequence data collected as part of the Sorcerer II Global Ocean Expedition (GOS)
also revealed a high abundance of viral sequences, representing
approximately 3% of the total predicted proteins in the 0.1–
0.8 μm size fraction of the plankton (Williamson et al., 2008). Viral
sequences revealed hundreds to thousands of viral genes, encoding various metabolic and cellular but mostly structural functions.
Quantitative analyses of viral genes of host origin confirmed the
viral nature of these sequences and suggested that significant portions of aquatic viral communities behave as reservoirs of such
genetic material. Distributional and phylogenetic analyses of these
host-derived viral sequences also suggested that viral acquisition
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of environmentally relevant genes of host origin is a more abundant and widespread phenomenon than previously appreciated.
The predominant viral sequences identified within microbial fractions originated from tailed bacteriophages and exhibited varying
global distributions according to viral family. The recruitment of
GOS viral sequence fragments against 27 complete aquatic viral
genomes revealed that only one reference bacteriophage genome
was highly abundant and was closely related, but not identical,
to the cyanobacterial myovirus P-SSM4 of Prochlorococcus hosts,
suggesting that this virus may influence the abundance, distribution, and diversity of one of the most dominant components
of small phytoplankton in oligotrophic oceans (Williamson et al.,
2008).
Overall, metagenomic analysis of viruses increasingly suggests
novel patterns of evolution, changes the existing ideas on the
composition of the virus world, and reveals novel groups of
viruses and virus-like agents (Kristensen et al., 2010). The gene
composition of marine DNA viromes is dramatically different
from that of known bacteriophages. The virome is dominated
by unknown genes, many of which might be contained within
virus-like entities such as gene transfer agents (GTA), which
are host DNA carrier particles (Lang et al., 2012). Analysis of
marine metagenomes thought to consist mostly of bacterial genes
revealed a variety of sequences homologous to conserved genes
of eukaryotic nucleocytoplasmic large DNA viruses, resulting in
the discovery of diverse members of previously undersampled
groups and suggesting the existence of new classes of virus-like
agents.
Unexpectedly, metagenomics of marine RNA viruses showed
that representatives of one superfamily of eukaryotic viruses, the
picorna-like viruses, dominate the RNA virome (Kristensen et al.,
2010). Marine RNA viruses are almost exclusively composed of
those that infect eukaryotes (Lang et al., 2009), primarily protists
(Culley et al., 2007). This was confirmed in a recent quantitative study where the comparison of the total mass of RNA
and DNA in viral fraction suggests that the abundance of RNA
viruses equaled or exceeded that of DNA viruses in coastal seawater (Steward et al., 2013). Similar findings were also reported
in freshwater systems (Roux et al., 2012). Because picorna-like
viruses have small genomes, they are at or below the detection limit of common fluorescence-based counting methods,
implying that protists contribute more to marine viral dynamics than one might expect based on their relatively low abundance.
Similarly, a recent metagenomic study from temperate and subtropical seawater has highlighted 129 genetically novel and distinct
viruses based on complete genome assemblages, all of which
were single-stranded DNA viruses mostly known as economically important pathogens of plants and animals (Labonté and
Suttle, 2013). The discovery of RNA and ssDNA viruses is a significant departure from the prevailing view of aquatic viruses which
are assumed to mostly contain double-stranded DNA and infect
bacteria. It is thus likely that we are missing a significant fraction of viruses in aquatic ecosystems, which probably is one of
the many reasons that may help explain the apparent discrepancy
between genome-derived and metagenome-derived diversity of
viruses. These reasons are highlighted in Ignacio-Espinoza et al.
(2013).
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Viruses of environmental microbes
ABUNDANCE, DISTRIBUTION, AND BIOGEOGRAPHY
Viruses were first suspected as abundant particles in the sea in
the late 1970s (Torrella and Morita, 1979), which was confirmed
one decade later with the discovery that 1 ml of sea water contains millions of viruses (Bergh et al., 1989). First estimates were
variable and inaccurate because they were based on manipulated
(i.e., ultracentrifuged) samples observed at high magnification
using transmission electron microscopy. More accurate and reproducible estimates were provided later using direct epifluorescent
microscopy or flow cytometry, yielding viral abundances that
exceed those of Bacteria and Archaea by an overall average of
about 15-fold (Bettarel et al., 2000). It is important to use fluorochrome dyes (e.g., SYBR Gold) that bind to ssDNA and ARN
viruses which have been proved, based on metagenomics, to be
much more abundant in natural aquatic systems than expected
(Lang et al., 2009; Holmfeldt et al., 2012). Furthermore, in this
context of methodological difficulties for accurate estimates of the
numerical abundance of natural viruses, it is important to stress
the recent discovery that DNA associated with membrane-derived
vesicles, gene transfer agents, or cell debris can produce fluorescent dots that can be confused with viruses (Forterre et al., 2013).
This targets a critical problem that needs to be bypassed in the
future because many bacterial species, including the dominant
marine cyanobacterium Prochlorococcus spp., release extracellular
vesicles which have roles in various processes (e.g., quorum sensing, virulence, horizontal gene transfers, etc.), and were recently
demonstrated to have a significant effect on carbon cycling in
marine ecosystems (Biller et al., 2014).
Viral abundance generally increases with the increasing productivity of aquatic ecosystems and, as a consequence, decreases
from freshwater to marine ecosystems, from costal to oceanic
zones, and from the surface to the bottom of the euphotic
layer (Sime-Ngando and Colombet, 2009). The abundance of
viruses in individual aquatic systems appears to be independent of salinity but related to the biomass of primary and
secondary producers, as well as to seasonal effects (Wilhelm
and Matteson, 2008). In the dark ocean (i.e., meso- and bathypelagic zones), where about 75% of prokaryotic biomass and
ca. 50% of prokaryotic carbon production in the world ocean
occur (Aristegui et al., 2009), high abundance of viruses was
observed (Parada et al., 2007). Similarly, two deep marine sediment studies from Ocean Drilling Project samplings in Saanich
Inlet, Canada (Bird et al., 2001), and on the Eastern margin of
the Porcubine Seabight (Middelboe et al., 2011) have reported
abundant viruses and prokaryotes in >100 m sediment cores
aged from 0 to 14,000 years and from 0.5 to 2 million years,
respectively. On a volumetric basis, viral abundances in sediments exceed 10–1000 times that in the water column, representing
active and mostly endemic components of benthic environments
(Danovaro et al., 2008), although visibly infected cells are often
scarce (Filippini et al., 2006). Because the relative abundances of
Archaea increase in the dark deep ocean and freshwater lakes,
viruses of Archaea are also expected to be abundant there,
as recently suggested by highly complex, diverse morphologies
observed in a deep-dark permanently anoxic freshwater lake, some
of which being putatively new for science (Borrel et al., 2012).
Thus, it is likely that the ecology of the deepest and benthic
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waters where eukaryotes are constrained by poor oxygen conditions is essentially driven by the dark viral loop (dissolved
organic matter–prokaryotes–viruses) processes (Colombet and
Sime-Ngando, 2012).
According to time, viral abundances fluctuate on diverse scales,
from minutes to years, often in association with prokaryotes,
which offer the major reservoir for hosts (Wommack and Colwell, 2000). Surprisingly, there is only one to two orders of
magnitude variation in virus abundance among systems (c. 100fold; 109 –1011 virus particles l−1 ) in spite of more than three
orders of magnitude variation in the planktonic biomass, as
one ranges from either coastal to offshore or from surface to
deep-water environments (Wilhelm and Matteson, 2008). This
may help to explain why the virus-to-prokaryote ratios (VBRs)
fluctuate substantially, with an overall increase from 3 to 10
in oligotrophic marine systems to 6–30 in productive freshwaters where the burst size (i.e., the number of viruses release per
lyzed host cell) and the contact and infection rates are generally
higher (Sime-Ngando and Colombet, 2009). The higher VBRs
in productive lakes may also reflect the increasing relative abundance of non-bacteriophage viruses along the trophic gradient
of aquatic systems (Bettarel et al., 2003). Virus abundances in
freshwaters appear to vary more strongly on seasonal scales than
in marine environments, especially in lakes that undergo pronounced seasonal cycles, although the linkages between seasonal
cycles and virus abundance remain unresolved in the absence of
long-term studies (Wilhelm and Matteson, 2008). Evidence that
viral abundance across oceans and lakes is driven by different factors was provided based on case studies, including bacterial and
cyanobacterial abundances, and chlorophyll-a concentration as
significant variables in lakes, bacterial and cyanobacterial abundances for coastal Pacific Ocean, and bacterial abundance and
chlorophyll-a concentration for coastal Arctic Ocean (Clasen et al.,
2008). However, this mainly concerns free-floating viruses in the
water column. Methodological progress based on confocal laser
scanning microscopy in combination with lectin and nucleic acid
staining has demonstrated that viruses trapped in organic aggregates are much more abundant than in the water column, ranging
from 108 to 1014 viruses l−1 . Organic aggregates and inorganic
particles appear to play a role of viral scavengers or reservoirs
rather than viral factories, and can enhance the growth rates of
free-living prokaryotic community. The problems and knowledge gaps in virus–particle interactions, and the related research
avenues and implications for water-column ecological processes
(e.g., microbial diversity, food web structure, biological pump,
biogeochemical cycles, etc.), are provided in an excellent review by
Weinbauer et al. (2009).
On a global scale, the forces that shape the biogeography of
viruses have received very little attention. It is of interest to search
for general patterns of microbial and viral biogeography because
general ecological theories, actually known solely from “macroscopic” or visible species (e.g., the positive relationship between
diversity and area sampled, or the negative one between local abundance and body size), will offer predictive tools in the context of
global change. Microorganisms and their viruses have long been
considered as ubiquitous, without a biogeography of any sort.
This is because their dispersal is thought to be unlimited due to
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small size, large absolute abundances and the formation of resistant or dormant stages. The ubiquity tenet for microorganisms
is the so-called Baas Becking statement “everything is everywhere,
but the environment selects” (Fontaneto, 2011). The assumption
that viruses are ubiquitous across habitats is currently being evaluated and some phages could be globally distributed, while others
could be unique and perhaps endemic to specific habitats (Roux
et al., 2012), primarily to extreme environments such as deserts
(Prigent et al., 2005; Prestel et al., 2008) or deep-dark permanently anoxic volcanic lake sediments (Borrel et al., 2012). It
was also extrapolated from metagenomic data that viral diversity could be high on a local scale but relatively limited globally,
and that viruses promote horizontal gene transfers by moving
between environments (Breitbart and Rohwer, 2005). Further
work is required to fully resolve and confirm the drivers of viral
large-scale distribution, in conjunction with the improvement of
taxonomy, methods, and sampling effort for both viruses and their
hosts.
LYTIC ACTIVITY, MICROBIAL MORTALITY, AND
BIOGEOCHEMICAL IMPLICATIONS
The diversity and the abundance of total viruses are not always
correlated to the lytic activity. Most free-occurring viruses are
considered infectious (Suttle, 2005). It is now well accepted that
lytic viruses represent one of the main causes of microbial mortality in aquatic systems (Figure 1). Based on the direct observation
of infected cells, viral-mediated mortality averages 10–50% of the
daily production of heterotrophic prokaryotes and approximately
equals the bacterivory from grazers in both fresh and marine
waters (Fuhrman and Noble, 1995; Pradeep Ram et al., 2005).
These values fluctuate largely (from zero to near 100%) depending mostly on the host availability (density and activity), although
physicochemical factors such as solar radiations (Wilhelm et al.,
1998), temperature (Pradeep Ram et al., 2005) or anoxia (Colombet et al., 2006) can impact the lytic activity of viruses. The
populations of lytic viruses ultimately depend on the availability of specific hosts, and could thus respond to the growth rate of
the most active hosts. This pattern has the strong feedback effect
of preventing species dominance and enhanced species cohabitation within microbial communities, i.e., the so-called phage kills
the winner hypothesis (Thingstad and Lignell, 1997). Viral lytic
activity was also demonstrated as uneven and heterogeneous for
different prokaryotic phenotypes and/or genotypes, a situation
which can shape the host diversity and community structure, and
thus exert a strong influence on the processes occurring in the
plankton food web dynamics (Pradeep Ram et al., 2010).
Because viruses kill microbial hosts which dominate the biological biomass in pelagic systems, including bacteria, archaea,
cyanobacteria, protists, and fungi as major partners, they have
an overwhelming effect, both directly and indirectly, on the
cycling of the major conservative elements (C, N, P, etc.) upon
which the food web dynamic is based (Fuhrman, 1999; Wilhelm
and Suttle, 1999). It was estimated that the absolute abundance
of oceanic viruses results in about 1029 infection events day−1 ,
causing the release of 108 –109 tons of carbon day−1 from the
living biological pool (Suttle, 2007). By exploding microbial
cells, lytic viruses are strong catalyzers of the transformation
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of living organisms to detrital and dissolved phases available to
non-infected microbes. This biogeochemical reaction increases
the retention time of organic matter and its respiration in the
water column and weakens the trophic efficiency of the food
web, but also provides nutrients (e.g., directly or indirectly from
mineralization and photodegradation of dissolved organic matter, DOM) to primary producers (Figure 1; Weinbauer, 2004).
For example, it has been shown that the iron contains in the
viral lysis products could fulfill the metabolic requirements of
marine phytoplankton (Poorvin et al., 2004). Primary production in the size fraction 2–200 nm can be depleted by about
half due to an increase in viral abundance as low as 20%. A
modeling exercise suggested that viral lysis of 50% of bacterial production could increase microbial respiration by 27%,
while decreasing the grazing efforts from protists and metazoan
zooplankton by 37 and 7%, respectively. When adding 7% of
viral-mediated loss of phytoplankton and 3% grazing of viruses
by phagotrophic flagellates, bacterial respiration increases to 33%
(Fuhrman, 1999).
The effects of lytic viruses thus directly affect DOM concentration but also its composition. For example, Lønborg et al. (2013)
recently demonstrated that viral lysate from an axenic culture of
Micromonas pusilla significantly change the DOM composition
by increasing the amounts of transparent exopolymer particles
(TEP), aromatic amino acids, and humic DOM, with an elevated
protein : humus ratio that suggested a higher contribution of
labile components in viral-produced DOM than in algal exudates. At the natural community level, these results suggested
that viral lysis could decrease the organic matter sedimentation and promotes respiration and nutrient retention whereas,
in contrast, the enhanced TEP production could stimulate particle aggregation and export out of the water column. Based
on metabolomics approach, Ankrah et al. (2014) demonstrated
that phage infection of Sulfitobacter sp. redirects 75% of nutrients into virions, and 71% of 82 intracellular metabolites were
significantly elevated in infected hosts, which also exhibited an
elevated metabolic activity compared to non-infected populations. In contrast, more than 70% of 56 compounds in viral
lysate decreased in concentration relative to uninfected controls,
suggesting that small, labile nutrients from viral lysis are utilized quickly by non-infected cells. Ankrah et al. (2014) conclude
that virus-infected cells are physiologically different from their
uninfected counterparts, a situation which can alter the ecosystem biogeochemistry. One of the intrinsic mechanisms for that is
the viral control of bacterial growth efficiency over a broad range
of values (from 0.1 to 70%) and the related patterns in carbon
and nutrient fluxes mediated by bacteria in pelagic environments
(Bonilla-Findji et al., 2008; Motegi et al., 2009; Pradeep Ram et al.,
2013).
There are many indirect ways that viruses can affect the
biogeochemical cycling. Lytic viruses may shape the global climate by inducing the release of dimethyl sulfide (C2 H6 S), a
gas known to influence cloud nucleation (Figure 1), which is
massively produced by major bloom-forming species such as
M. pusilla, E. huxleyi, and Phaeocystis pouchetii (Evans et al.,
2007). Viral lysis of microorganisms in sinking aggregates could
also decrease the sinking rate by alleviating the aggregates via
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Viruses of environmental microbes
release of trapped dissolved and colloidal materials. Alternatively,
virus-infected cells could sink faster compared to non-infected
cells (Lawrence and Suttle, 2004), contributing to the export
of microbial cells downwards (Figure 1). In addition, viral
lysis products contain polymers that can increase gel formation
and affect the biological, physicochemical and optical properties
of the sea water (Uitz et al., 2010), for example by generating aggregates and enhancing the departure of organic material
from the euphotic zone (Lønborg et al., 2013). This can influence the amount of carbon exported to the deep ocean by the
so-called biological pump, but also the Redfield stoichiometry of the water column, because the export of nutrients other
than carbon needs to be balanced by new inputs (Mari et al.,
2005). Hence, highly labile N- and P-nutrients contained in
nucleic acids and amino acids, for example, will be used rapidly
and retained in the euphotic layer, while the sinking particles
will be abnormally rich in carbon, thereby increasing the efficiency of the oceanic biological pump activity (Figure 1; Suttle,
2007).
LYSOGENY: A SURVIVAL STRATEGY FOR VIRUSES
One of the key explanations for the omnipresence of viruses in
natural ecosystems is undoubtedly through the existence of several lifestyles, of which two major pathways, namely lysis and
lysogeny, are prevalent in aquatic systems (Pradeep Ram and
Sime-Ngando, 2010). Lytic infections are by far the best studied of the virus–host interactions. Lysogenic activity has been
less studied in aquatic environments where the temperate phage
can alternatively integrate into the host genome as prophage.
Prophages can be stable in their host for long periods of time,
from months to years, with low probability of bacteriophages being
released by spontaneous lysis (Paul, 2008). Examinations of natural prokaryotic communities inducible with a mutagenic agent
(e.g., mitomycin C) have suggested that the fraction of lysogenic
bacteria is typically <50% (range 0–100%) of the total abundance
in marine environments. In freshwaters, these values fluctuate
from 0 to 16% in temperate and tropical lakes, and from 0 to 73%
in Antarctic lakes (Sime-Ngando and Colombet, 2009).
Lysogenic conversion has been described as a means of survival for viral populations that are threatened by poor host cell
abundance and therefore cannot sustain population numbers
through lytic infection alone (Stewart and Levin, 1984; Palesse
et al., 2014). This situation occurs when the prokaryote abundance drops under a minimal threshold level, typically under
about 105 cells ml−1 (Pradeep Ram et al., 2005). Microcosm
experiments have recently demonstrated that nutrient addition in
freshwater samples stimulates lytic viruses via enhanced growth
rate of prokaryotes and, when limiting, rather promote lysogenic conversion (Pradeep Ram and Sime-Ngando, 2010). This
finding was considered an explanation why lytic and lysogenic
activities are often antagonistically correlated, supporting the idea
that lysogeny may represent a maintenance strategy for viruses in
harsh nutrient/host conditions which appeared as major instigators of the trade-off between the two viral lifestyles (Colombet
et al., 2006). Both viral life cycles are thus apparently regulated
by distinct factors, including environmental parameters (primarily resources for hosts) and host physiology for lytic cycle but
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mainly host physiology for lysogenic cycle (Maurice et al., 2013;
Palesse et al., 2014). It was also shown that the latter cycle is
prevalent within the phylogenetic groups that dominate the whole
bacterial community composition at a given time (Maurice et al.,
2011).
VIRUSES AND MICROBIAL DIVERSITY
Viruses can impact microbial diversity and force diversification mechanisms toward host-cell evolution in two major ways
(Figure 1). The first major way includes the direct effects of the
intrinsic activities of viruses: (i) keep in check competitive dominants (i.e., lytic viruses), (ii) affect the metabolic properties of
host cells which can acquire immunity to superinfections and new
phenotypic and genotypic traits such as production of toxins (i.e.,
temperate phage conversion), and (iii) transfer both viral and host
genes between species (transduction, GTA), thereby influencing
speciation. The second major way comprises the indirect effects
of viral activities such as (iv) the structuring effects of lysis products on species composition and richness, (v) the sustenance of
the amount of information encoded in genomes that may favor
horizontal gene transfer mechanisms, and (vi) the effects of physiological mechanisms involved in the resistance of host against
viruses, through the host-pathogen arms race (Figure 1). Together
with (i) the high abundance and broad geographical distribution
of viruses and viral sequences within microbial fractions, and
(ii) the prevalence of genes among typical viral sequences that
encode microbial physiological functions, the above-mentioned
effects establish environmental viruses as strong vectors that generate genetic variability of aquatic microorganisms and drive both
ecological functions and evolutionary changes (Weinbauer and
Rassoulzadegan, 2004).
VIRAL LYSIS PRODUCTS SHAPE EVOLUTIONARY TRANSITION, GLOBAL
CARBON CYCLING AND THE DIVERSIFICATION OF MICROBIAL
COMMUNITIES
Some viral groups such as the Caudovirales, the tailed doublestranded DNA phages, are probably older than the separation of
life into the three now recognized domains of life (Ackermann,
1999; Hendrix, 1999). This suggests that, before the occurrence
of eukaryotic grazers such as flagellates and ciliates, viruses were
probably the main predators of cells in the prokaryotic world, and
played a major role in the sophisticated forces (dispersal, competition, adaptive radiation, etc.) that shape biogeography and
evolution. In contemporaneous marine systems, it was estimated
that between 6% and 26% of the photosynthetically fixed carbon is channeled or “shunted” to the DOM pool by viral lysis
of cells at all trophic levels (Wilhelm and Suttle, 1999). The carbon stored in the oceanic DOM pool equals that in atmospheric
CO2 (Hedges, 1992), suggesting that viral infection of marine
prokaryotes and phytoplankton has an influence not only on global
carbon cycling and climate but also on the microbial composition
and community structure. A host density-dependent model, i.e.,
“the phage kills the winner” model (Thingstad and Lignell, 1997;
Thingstad, 2000), was proposed based on the assumption that
lytic viruses are highly specific to their host cells, at least at the
species level. In this model, by eliminating the most competitive
strains for resource acquisition, lytic viruses prevent dominance
Frontiers in Microbiology | Aquatic Microbiology
Viruses of environmental microbes
and increase niche availability for species-coexistence (Figure 1).
This was recently tested by Motegi et al. (2013) who provide
experimental data showing that viruses prevent the prevalence of
taxa that were competitively superior in phosphate-replete conditions in NW Mediterranean surface waters. In addition, these
authors obtained a statistically robust dome-shaped response of
bacterial diversity to viral (VP) to bacterial (BP) production ratio,
with significantly high bacterial diversity at intermediate VP:BP,
corroborating the prediction from the general model that species
diversity is maximized when productivity and disturbance are
balanced.
LYSOGENS AND THE EFFECTS OF THEIR MUTUALISTIC VIRUSES
Virus-host interactions range in a gradient from true non-lethal
parasitism (i.e., chronic infection) to fatal lytic infection, with
intermediate mutualistic lifestyles (lysogeny, pseudolysogeny)
where viral genomes stay within the host and confer new metabolic
traits which can increase the fitness of the immune host but also
the survival of the phage (Figure 1). A spectacular case for such
a phage conversion is the finding that cholera infection is due
to a lysogenic strain of the Vibrio cholerae bacterium. Since the
toxin is encoded in the genome of the prophage and is not part
of the host genome, non-lysogenic cells do not cause cholera
(Weinbauer, 2004). In addition, it was recently shown that the
type VI secretion systems in V. cholerae are virulence-associated
proteins that are evolutionarily related to components of bacteriophage tails (Basler et al., 2012). Prophage induction events
can change the bacterial community structure by increasing the
diversity and richness of natural bacterial populations (Hewson
and Fuhrman, 2007). For example, phage conversion can increase
the fitness of cells (Edlin et al., 1975; Lin et al., 1977), which
could influence community composition by allowing for the survival or dominance of such converted cells. Host immunization
against infection by homologous phages and phage conversion
are ways in which phages can influence microbial diversity in
natural environments. More generally, it is likely that all living
cells could contain active prophages in their genome. On average, 2.6 prophages have been detected per free-living bacterial
species (Lawrence et al., 2002), and a number of bacterial genomes
contain between 3 and 10% of DNA prophages (Brüssow and
Hendrix, 2002). It was recently demonstrated that genes captured
from ancestral retroviruses have been pivotal in the evolutionary acquisition of the key process through which most of the
maternofoetal exchanges take place in placenta development in
mammalian species (Dupressoir et al., 2009). This highlights the
potential of phages as key players in the evolution and maintenance
of living things.
LATERAL GENE TRANSFERS (LGTs) BY TRANSDUCTION: VIROMES ARE
HUGE RESERVOIRS OF VIRALLY ENCODED HOST GENES THAT ARE
CANDIDATES FOR LGTs
One of the most surprising findings of whole-genome sequencing is the enormous extent of lateral gene transfers. LGTs refer
to the gene material exchanges between organisms that happen
independently of reproduction (i.e., vertical gene transfers). General mechanisms include transformation (gene transfer by uptake
of free genetic materials), conjugation (direct gene transfer from
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cell-to-cell contact), the activity of GTAs, and transduction, where
viruses are the main vectors that move nucleic materials from one
cell to another (Figure 1). The transduction frequency in natural
waters ranges from 10−8 to 10−5 per virus, and they might be
up to 100 transductants l−1 day−1 (Jiang and Paul, 1998). An
extrapolation exercise suggests that as many as 1024 genes are
moved by transduction from viruses to hosts each year in the
world’s ocean (Rohwer and Thurber, 2009). This is considered an
underestimate because of the action of the host DNA carrier GTAs
(primarily in the α-Proteobacteria order Rhodobacterales) which
are injected into recipient cells, providing a more efficient form of
transduction (Lang et al., 2012). Although transduction is a random process, viruses can genetically alter microbial populations
through lysogeny and transduction, and affect the flow of genetic
information in aquatic ecosystems.
Large-scale metagenomics has shown that viruses contain
diverse genes of interest, including virulence genes such as the
cholera toxin genes, respiration, nucleic-acid, carbohydrate and
protein metabolism genes, as well as genes involved in vitamin
and co-factor synthesis, in stress response, and in motility and
chemotaxis, which are more common in viromes (metagenomes of
viruses) than in their corresponding microbiomes (metagenomes
of microbes; Rohwer and Thurber, 2009). Microbes that take up
these genes increase their competitive ability and extend their ecological niches (Figure 1). More interestingly, virally encoded host
genes also include crucial photosynthetic genetic elements present
in cyanophage genomes, which can be used to maintain the targeted function in dead hosts and accomplish the lytic cycle, and
can be transferred between hosts as well (Lindell et al., 2005).
About 10% of total global photosynthesis could be carried out
as a result of phage genes originally from phages (Rohwer and
Thurber, 2009). Given the prevalence of phage-encoded biological functions and the occurrence of recombination between phage
and host genes, phage populations are thus expected to serve as
gene reservoirs that contribute to niche partitioning of microbial
species in aquatic ecosystems. Gene transfers by transduction may
also represent an important mechanism for gene evolution in natural environments, and bacteriophage transduction could play an
important role in contributing to the genetic diversity of microbial
populations.
INDIRECT EFFECTS OF VIRUSES ON MICROBIAL DIVERSITY
The mass release of lytically infected cell contents can change
the composition and the bioavailability of ambient organic substrates and nutrients, which are well known as key factors affecting
the microbial composition and community structure (Figure 1).
It has been shown that the presence of grazers in phosphoruslimited microcosms appeared to be a stimulating factor for
prokaryotic growth and lytic viral proliferation, with a significant increase in the minor bacterial phylotypes as a consequence
of the reduction of resource competition in prokaryotic assemblages (Sime-Ngando and Pradeep Ram, 2005). Prokaryotic phyla
belonging to Bacteria, β-Proteobacteria and α-Proteobacteria
responded significantly to lysis products, while Archaea and
Cytophaga-Flavobacterium (now known as Bacteroidetes) rather
changed their community size structure towards grazing-resistant
forms (Pradeep Ram and Sime-Ngando, 2008, 2014). In general,
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Viruses of environmental microbes
the presence of grazers is a stimulating factor for prokaryotic
growth and viral proliferation in the plankton, probably through
nutrient regeneration process that increases niche availability and
enhances prokaryotic diversity (Pradeep Ram and Sime-Ngando,
2008, 2014). The relative abundance, production and species
richness of some bacterial phyla such as Flectobacillus or Actinobacteria increase more in the presence of both viruses and
grazers that when only one of the consumers is present (Simek
et al., 2007). Although, Weinbauer et al. (2007) reported both
synergistic and antagonistic effects of viral lysis and protistan
grazing activities on bacterial biomass, production, and diversity,
and considered these effects as the result of group- or speciesspecific competition for prey and hosts, and the fact that both
types of predators produce organic matter that potentially fuel
growth.
Lysis products can contain phage-borne enzymes which can
kill cells and also influence microbial composition (Fuhrman and
Noble, 2000). Lysis products also include free genetic materials that
can increase the amount of information encoded in genomes in
the water column, and favor horizontal gene transfer mechanisms
such as conjugation, transformation or genetic recombination
(Weinbauer, 2004). Because all of the direct and indirect roles
of viruses in LGTs ultimately result in “novel” genetic materials
and information moving into the host cell, there are strong interactions between lateral and vertical gene transfer mechanisms. The
acquisition of new genes can affect the genome size and the generation time of host communities and, perhaps more importantly,
can move the strain or species barriers in microbial communities
(Weinbauer and Rassoulzadegan, 2004). This can also affect the
metabolisms and physiology of the host and, hence, their susceptibility to viral infection can be weakened up to a total resistance
against viral infections. We are thus in the presence of an effective
host-pathogen arms race for survival and coexistence, where host
resistance is crucial for offspring and maintenance (Figure 1).
REISTANCE OF VIRAL HOST CELLS
Hosts and pathogens persist in the environment mainly through a
molecular arms race between competing hosts and viruses where,
as already discussed, viruses can affect their host in various ways,
ranging from the enhancement of the host fitness and metabolic
performances to mortality. Because parasites and pathogens tend
to have shorter generation time than their hosts, they should evolve
more rapidly and maintain advantage in the evolutionary race
between defense and counter-defense. The paradox here is how do
victim species survive and even thrive in the face of a continuous
onslaught of more rapidly evolving enemies? One of the explanations is that the physiological, mechanical and behavioral costs of
defense are lower compared to the cost of attack (Gilman et al.,
2012). The viral host communities can thus respond to the pressure from their parasites and develop a sophisticated resistance
shield, specific to each step in the viral cycle, but suffer from the
cost of resistance which mainly includes a decrease in the fitness
and growth rate or adaptive responses (review in Thomas et al.,
2011), such as the production of proteins or extracellular matrices
that mask the phage receptor (Bohannan and Lenski, 2000).
Viral receptors on the cell surface are complex families of proteins, carbohydrates or lipids, which serve normal physiological
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functions of the cells but are hijacked by viruses for their adsorption. Host cell mutations and resistance to viral adsorption grossly
include modifications of the receptor structure, alterations of
receptor accessibility, decreases in the number of receptors, or
loss of receptor sites. A spectacular example of host surface modification is the so-called “Cheshire Cat” strategy where the resistant
haploid phase of the algal haptophyte E. huxleyi does not calcify
and is “invisible” to viruses, in contrast to the susceptible diploid
phase (Frada et al., 2008). Similarly, it was shown that colonial
forms of the algal prymnesiophyte P. pouchetii are resistant to
viruses because they are surrounded by an “outer skin,” in contrast
to individual cells (Brussaard et al., 2007; Jacobsen et al., 2007).
Brussaard et al. (2005) demonstrated that the morphology (solitary versus colonial) of the prymnesiophyte Phaeocystis globosa
differently regulate viral control of P. globosa bloom formation,
depending on irradiance, nutrient, and grazing regimes. A modeling exercise suggested that the enhanced growth rates, the low
viral infection rate, and the low grazing rate on cells in colonies,
as compared to free-living single cells of P. globosa, result in a
massive blooming of P. globosa colonies. When the controlling
nutrient becomes depleted, the colonies disintegrate and liberate
single colonial cells that are subject to high rates of viral infection
and grazing (Ruardji et al., 2005). It is thus likely that cell hosts
embedded in colonies are more resistant to viral infection than
free-living cell hosts.
The host mechanisms of blocking viral replication and entry
remain relatively unknown in marine organisms. Stolt and Zillig (1994) reported a prophage-encoded gene (rep) in the marine
archaea Halobacterium salinarum able to protect cells from viral
infection. Tomaru et al. (2009) have shown that viral genome
replication in resistant cells of the dinoflagellate Heterocapsa circularisquama is repressed. Bacteria, cyanobacteria, and archaea are
known to prevent viral replication by the acquisition of immune
systems consisting of short fragments of foreign nucleic acids into
clustered interspaced short palindromic repeats (CRISPRs) in their
genomes. CRISPR spacers have homologies with mobile genetic
elements such as bacteriophages and plasmids, and those identical to phage sequences provide resistance against viral infection
(Thomas et al., 2011). Overall, the various effects of viruses force
costly resistance mechanisms in their host communities, where the
co-existence of sensitive and resistant host cells is likely a result of a
trade-off between competitive ability and mortality. Lennon et al.
(2007) provided a nice example of the ability of marine cyanobacteria Synechococcus to evolve resistance with fitness costs associated
with the identity of a few particular viruses, suggesting that variability in fitness costs associated with viral resistance can structure
microbial communities and regulate biogeochemical cycles.
VIRUSES AS CELL PARTNERS AND CELL MANIPULATORS
Although viruses are most often studied as pathogens, many are
beneficial to their hosts, providing essential functions in some
cases and beneficial functions in others. For example, many
pathogenic bacteria produce a wide range of virulence factors that
help them to infect their hosts. There are numerous examples of
such virulence factors that are expressed not from the bacterial
genome but from a phage genome, such as diphtheria, Shiga, and
cholera toxins (Brüssow et al., 2004). The nuclei of dinoflagellates
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Viruses of environmental microbes
contain permanently condensed, liquid crystalline chromosomes
that seemingly lack histone proteins, and contain remarkably large
genomes. The molecular basis for this organization was recently
provided by Gornik et al. (2012) who discovered that histone
proteins were replaced, during evolutionary events, by a novel,
dominant family of nuclear proteins (called DVNPs, dinoflagellate/viral nucleoproteins) that is only found in dinoflagellates and,
surprisingly, in a family of large algal viruses, the Phycodnaviridae. The authors concluded that gain of a major novel family of
nucleoproteins from an algal virus occurred early in dinoflagellate
evolution and coincided with rapid and dramatic reorganization
of the dinoflagellate nucleus.
Although few examples for viral manipulators were reported
in the microbial world, studies from eukaryotes have shown that
some viruses are essential for the survival of their hosts; others give
their hosts a fighting edge in the competitive world of nature, while
some others have been associated with their hosts for so long that
the line between host and virus has become blurred (Roossinck,
2011). Some virologists think that modern genomes are essentially
remnants of ancient viruses. Intact and fragmented retroviruses
are found in the genomes of almost all eukaryotes. Approximately
8% of the human genome is derived from retroviruses (Lander
et al., 2001), and this percentage increases dramatically if other
mobile genetic elements are included (Kazazian, 2004). At least
some endogenous retroviruses encode functional genes and are
thought to be involved in major evolutionary leaps. For example, the evolution of placental mammals probably occurred after
the endogenization of a retrovirus. Retroviral envelope proteins
cause fusion of cell membranes, a process that not only allows the
invasion of oncogenic viruses but also is required for the development of the placental syncytium, an essential part of the barrier
that prevents maternal antigens and antibodies getting into the
fetal bloodstream and provoking abortion (Dunlap et al., 2006).
All living things thus have something of viruses in their genomes,
some of which may be beneficial to their fitness and evolution.
Furthermore, recent researches are increasingly demonstrating the ecological and evolutionary importance of viruses of
symbiotic organisms within their hosts, in complex tripartite
interactions. A famous example is the existence of an endogenous virus that (i) alter the life story of the sea photosynthetic
slugs Elysia chlorotica by possibly synchronizing the lifetime of
the stolen chloroplasts to that of the slugs, (ii) and is probably the vector of the horizontal gene transfer between the slug
and the chloroplasts that provides 80–90% of the genes necessary for photosynthesis (Rohwer and Thurber, 2009). However,
recent analysis of the genome of E. chlorotica egg DNA provides
no clear evidence of horizontal gene transfer into the germ line of
this kleptoplastic mollus, and suggests that algal nuclear genes
or gene fragments are present in the adult slug (Bhattacharya
et al., 2013). Polydnaviruses (PDVs) are viruses associated with
wasp species that parasitize lepidopteran larvae. PDV particles are
injected along with the eggs of the wasp into the lepidopteran larvae (or eggs) and express proteins that interfere with host immune
defenses, development, and physiology; this interference enables
wasp larvae to survive and develop within the host. It was recently
shown that the PDV particles are well conserved in the braconid
wasp ovaries and originated from the integration of nudivirus
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machinery into the genome of an ancestral wasp about 100 million years ago. Bézier et al. (2009) found that nudiviral genes
themselves are no longer packaged but are actively transcribed
and produce particles used to deliver genes essential for successful parasitism in lepidopteran hosts. Recent works in the field of
entomology have revealed tripartite associations between insects,
bacteria, and phages, where phages control bacteria which affect
the physiology and ecology of the host. Overall, it is clear that
viruses are key players in complex symbiotic associations between
animals and their sequestered plant chloroplasts, parasitoid insects
and their insect hosts, and between bacterial pathogens and their
insect hosts. This is an overlooked role of environmental viruses
that probably occurs in all habitats where viruses vector important traits, such as defense against or sensibility to parasitoids,
within and among symbionts of animal and probably plant host
lineages.
CONCLUDING REMARKS
Aquatic viral ecology is a relatively recent discipline, in increasing
development. Viruses are omnipresent in aquatic environments,
including the most extreme and worst biotopes, where they often
represent the most abundant biological entity. Because all types of
cell in the three domains of life have their specific viruses and offer
ecological niches to different viral lifestyles, viruses are considered
the greatest reservoir of the uncharacterized biological diversity
on the earth, which is being probed and described at an increasingly rapid rate, almost exclusively with molecular sequence data.
For example, Hewson et al. (2013) recently used a metagenomic
approach to identify circular, single-stranded DNA viruses that
may be involved in the seasonal dynamics of Daphnia spp. in
Oneida and Cayuga lakes (upstate New York). Because Daphnia
plays a critical role in many lake ecosystems, such viruses may have
important effects on herbivory and thus carbon flow through the
lake ecosystem.
Our conceptual understanding of the function and regulation
of aquatic ecosystems, from microbial to global biogeochemical
processes, has changed with the study of viruses. Viral-mediated
prokaryotic mortality roughly equals bacterivory from protists,
which is a significant departure from the traditional view that
predation and resource availability are the main factors controlling prokaryotic abundance and production in pelagic systems.
Viruses influence both the retention and the export of organic
matter in the pelagic realms. Given the prevalence of phageencoded biological functions within host cells and the occurrence
of recombination between phage and host genes, phage populations serve as gene reservoirs that contribute to niche partitioning
of microbial species in aquatic ecosystems. Viral-mediated gene
transfers include diverse mechanisms (transduction, transformation, conjugation, and recombination) that are known to affect
gene evolution in the marine environment. It is thus clear that
most of the viruses are not pathogens but mutualistic cell partners that provide helper functions. The discovery of giant viruses
of eukaryotes (absent in this review) which encode trademark
cellular functions has weakened the gap between inert and living things. Overall, studies in aquatic viral ecology are sources
of novel knowledge related to the biodiversity of living things,
the functioning of ecosystems, and the evolution of the cellular
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Viruses of environmental microbes
world. The future challenge is to extend viral ecology studies
(i) to all biotopes in the biosphere such as lakes, rivers, underground waters, soils, clouds, air, etc., (ii) to all living organisms
which are susceptible to viral attacks (e.g., zooplankton, Archaea
etc.), and (iii) the related functions, some of which are crucial to
global change (e.g., consumption and production of greenhouse
gas such as methane etc.), and (iv) to multi-partner symbioses and
their effects on the food web dynamics and host maintenance and
adaptive evolution.
More generally, next-generation sequencing technologies are
increasingly revealing that microbial taxa likely to be parasites or
symbionts are probably much more prevalent and diverse than
previously thought. Every well-studied free-living species has parasites; parasites themselves can be parasitized. As a rule of thumb,
there is an estimated four parasitic species for any given host,
and the better a host is studied the more parasites are known to
infect it. Therefore, parasites and other symbionts should represent a very large number of species and may far outnumber those
with “free-living” lifestyles. Paradoxically, free-living hosts, which
form the bulk of our knowledge of biology, may be a minority. Microbial parasites offer good experimental models because
they are typically characterized by their small size, short generation time, and high rates of reproduction, with simple life cycle
occurring generally within a single host. They are diverse and
ubiquitous in aquatic ecosystems, comprising viruses, prokaryotes, and eukaryotes. Extensive studies on all aspects of parasites
and other symbionts in aquatic microbial ecology are warranted,
including method development, life cycle, interactions with hosts
and competing microbes, coevolution, effects on food webs, and
biogeochemical cycles. I believe that including viruses in the more
complex world of parasites and symbionts is promising for biology
and ecology in the future.
ACKNOWLEDGMENTS
The author is particularly indebted to students and postdocs of mine (cf. download the list at http://www.lmge.univbpclermont.fr/spip.php?rubrique100), particularly to those who
have worked on the topic reviewed herein: Yvan Bettarel, Jonathan
Colombet, Mylène Hugoni, Tomohiro Mochizuki, Stéphanie
Palesse, Angia Sriram Pradeep Ram, and Agnès Robin. Two reviewers provided thorough and excellent reviews of this study. It was
supported by the French “ANR Programme Blanc,” ROME Project
(ANR 12 BSV7 0019 01).
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Conflict of Interest Statement: The author declares that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 22 January 2014; accepted: 25 June 2014; published online: 24 July 2014.
Citation: Sime-Ngando T (2014) Environmental bacteriophages: viruses of microbes in
aquatic ecosystems. Front. Microbiol. 5:355. doi: 10.3389/fmicb.2014.00355
This article was submitted to Aquatic Microbiology, a section of the journal Frontiers
in Microbiology.
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